*3.1. Whole-Genome Sequence Diversity Data of K133AS-R Compared to K133WT*

Comparative WGS data analysis of both in vitro-generated artemisinin-resistant parasite (K133AS-R) and the wild-type field isolate (K133WT) was performed to decipher the mechanisms responsible for drug resistance. Detailed analysis of SNPs and insertion-deletion mutations (InDels) was performed for K133WT and K133AS-R isolates relative to the *L. donovani* reference using GATK's Haplotype Caller (HC) [59]. WGS data analysis of K133WT showed a higher number of upstream gene variants followed by intergenic region and missense gene variants. Out of a total of 341 gene variants, 191 SNPs and 150 InDels were observed (Figure 1A). The maximum number of SNPs was observed on chromosome number 34, while no SNP was observed on chromosome number 5, 9, 11, 14, 21, and 26 out of a total of 36 chromosomes in *Leishmania*. Amongst the total InDels, 114 nucleotide insertions and 36 nucleotide deletions were observed.

The artemisinin-resistant parasite generated in vitro under drug selection pressure (K133AS-R) showed a higher number of upstream gene variants followed by intergenic region gene variants. Out of a total of 477 gene variants, 240 SNPs and 237 InDels were observed (Figure 1A).

**Figure 1.** *Cont*.

**Figure 1.** Comparative Analysis of Single Nucleotide Polymorphisms (SNPs) present in K133AS-R with K133WT. (**A**) Venn diagram showing the unique genes present in K133WT and K133AS-R. (**B**) Comparative SNP density analysis of K133WT vs. K133AS-R (**C**) Pie chart showing the percentage of different gene variants present in K133WT and K133AS-R.

The maximum number of SNPs was observed on chromosome number 31, while no SNPs were found on chromosome number 14 and 26, which is a common observation among K133WT and K133AS-R (Figure 1B). Among InDels, 173 nucleotide insertions and 64 nucleotide deletions were observed. Unique gene variants were also observed among K133WT and K133AS-R. In K133WT, the unique gene variants identified were upstream variant 254 (74.48%), downstream 23 (6.74%), missense 27 (7.9%), frameshift 7 (2.05%), and intergenic region gene variants 27 (7.9%), while other variations included disruptive / conservative in-frame insertions, one each and one conservative in-frame deletion, which is 0.3% of the total gene variation observed. In case of K133AS-R, the unique gene variants observed were 357 (74.84%) in upstream, 45 (9.43%) in downstream, 47 (9.85%) in intergenic region, 16 (3.35%) missense variant, 11 (2.05%) frameshift variants, and one stop-lost splice variant (0.21%) (Figure 1C). Insertions were observed to be the highest in the genome followed by transition, transversion, and deletion.
